Use Of Supercritical Fluid Chromatography Research Articles

A Dual Column pH Switchable Water Stationary Phase System for Separation Control in Supercritical Fluid Chromatography.

A dual column system comprised of a pH switchable water stationary phase column and a conventional non-polar capillary column is introduced for use in Supercritical Fluid Chromatography (SFC). By removing or adding NH4OH to the system hydration source, the water stationary phase pH can be rapidly switched between acidic (measured at pH∼3) and basic (measured at pH∼9) in seconds, while the operating character of the conventional column is unchanged. This switch modulates the velocity of ionizable analytes about 20-fold in the system, whereas non-ionizable analytes are not affected. In this way, the retention time of acids and/or bases can be reproducibly altered (<1% RSD; n = 3) in SFC separations. As a result, analyte selectivity and resolution can be readily controlled during analyses. For example, a selectivity reversal (alpha from 0.4 to 1.6) and a resolution increase (from 0 to 13) are demonstrated. Rapid stationary phase pH switching also allows multiple acids, bases, and/or neutral analytes to be determined simultaneously. Applications demonstrate that this method can greatly simplify complex mixture analysis in SFC by helping to separate target analytes from interfering matrix components.

A novel switchable water stationary phase for supercritical fluid chromatography

A novel pH switchable water stationary phase is presented for use in supercritical fluid chromatography (SFC). By adding NH4OH to the water coating and system hydration, changes in CO2 pressure and temperature allow a wide range of stationary phase pH conditions (∼3–9) to be achieved, which impact analyte retention properties. For example, 100 atm and 50 °C produces an acidic water stationary phase (pH near 4.0) where octanoic acid readily elutes while the base caffeine does not. Conversely, at 80 atm and 120 °C a basic water stationary phase (pH near 8.0) is obtained and the opposite occurs. Further, under constant pressure and temperature conditions, simply adding or removing NH4OH from the system is also found to readily allow switching between the basic and acidic water stationary phase modes and demonstrates control over ionizable analyte elution. For instance, hexanoic acid elution is near 40 times more delayed on a basic water stationary phase and, as such, it can be eluted at later points in time as desired by removing the NH4OH and switching to an acidic stationary phase. Experiments indicate that stationary phase pH switching occurs uniformly across the 15 m column length within about 18 s and that analyte retention times are very reproducible upon performing a switch (1.4% RSD; n = 3). Results demonstrate the selectivity factor between acidic and neutral analytes can be reversed and increased about 35 times, while in other trials resolution also similarly increased near 40-fold. By rapidly switching the stationary phase pH back and forth between acidic and basic modes, the selectivity between ionizable analytes could also be increased as desired. Various applications with the system show that it can vastly increase the separation between target analytes and matrix components as required by the dynamics of a particular separation.

Separation of diarylethene-based photoswitchable isomeric compounds by high-performance liquid chromatography and supercritical fluid chromatography.

Diarylethene-based photoswitches have become very popular over the last few decades for potential applications in chemistry, materials science, and biotechnology due to their unique physical and chemical properties. We report the isomeric separation of a diarylethene-based photoswitchable compound using high-performance liquid chromatography. The separated isomers were characterized by ultraviolet-visible spectroscopy and mass spectrometry confirmed the isomeric nature of the compounds. The isomers were purified by preparative high-performance liquid chromatography, providing fractionated samples to study the isomers individually. A total amount of 13mg of an isomer of interest was fractionated from a solution of 0.4mg/ml of the isomeric mixture. Because the preparative high-performance liquid chromatographic method required large quantities of solvent, we explored the use of supercritical fluid chromatography as an alternative separation mode which, to the best of our knowledge, is the first time this technique is used to separate diarylethene-based photoswitchable compounds. Supercritical fluid chromatography provided faster analysis times while maintaining sufficient baseline resolution for the separated compounds and consuming less organic solvent in the mobile phase compared to high-performance liquid chromatography. It is proposed that the supercritical fluid chromatographic method be upscaled and used in future fractionation of the diarylethene isomeric compounds, becoming a more environmentally benign approach for compound purification.

Ultrafast simultaneous chiral analysis of native amino acid enantiomers using supercritical fluid chromatography/tandem mass spectrometry

In the chiral separation of amino acids, liquid chromatography has been mainly used because of the physicochemical properties of the analytes. To date, only few reports of the use of supercritical fluid chromatography (SFC) for the analysis of chiral amino acids exist, and there is much room for improvement in terms of the number of measurable amino acids, peak shape, and analysis time. In this study, we developed a novel method for the chiral analysis of native amino acids using a system combining SFC and tandem mass spectrometry. Specifically, the separation of amino acid enantiomers was investigated using a CROWNPAK CR-I(+) column with a chiral stationary phase of optically active crown ether. Methanol/water mobile phase with trifluoroacetic acid as a modifier based on supercritical carbon dioxide (CO2) was used. At a low modifier concentration of 30% for the separation of hydrophilic compounds, 18 proteinogenic amino acid enantiomers except glycine and proline were successfully separated with resolution (Rs) = 1.96–33.62 within 6.5 min. In attempt to shorten the analysis time, the flow rate was increased; using a CO2/modifier ratio of 60/40 at a flow rate of 3 mL/min, ultrafast chromatography of 17 amino acid enantiomers, except histidine, was achieved with retention time ≤ 1 min and resolution ≥ 1.5. The developed ultrafast chiral separation method was verified by analyzing a commercially available black vinegar, which detected eight kinds of d-amino acids. The present method has thus confirmed to be successful and practical in terms of both analyte coverage and throughput.

Stereoselective metabolism of chloramphenicol by bacteria isolated from wastewater, and the importance of stereochemistry in environmental risk assessments for antibiotics

Wastewater treatment plants have been highlighted as a potential hotspot for the development and spread of antibiotic resistance. Although antibiotic resistant bacteria in wastewater present a public health threat, it is also possible that these bacteria play an important role in the bioremediation through the metabolism of antibiotics before they reach the wider environment. Here we address this possibility with a particular emphasis on stereochemistry using a combination of microbiology and analytical chemistry tools including the use of supercritical-fluid chromatography coupled with mass spectrometry for chiral analysis and high-resolution mass spectrometry to investigate metabolites. Due to the complexities around chiral analysis the antibiotic chloramphenicol was used as a proof of concept to demonstrate stereoselective metabolism due to its relatively simple chemical structure and availability over the counter in the U.K. The results presented here demonstrate the chloramphenicol can be stereoselectively transformed by the chloramphenicol acetyltransferase enzyme with the orientation around the first stereocentre being key for this process, meaning that accumulation of two isomers may occur within the environment with potential impacts on ecotoxicity and emergence of bacterial antibiotic resistance within the environment.

Fast analysis of alkaloids from different parts of Mahonia bealei (Fort.) Carr. studied for their anti-Alzheimer's activity using supercritical fluid chromatography.

In this study, a rapid and highly efficient method was developed for the separation of eight isoquinoline alkaloids using supercritical fluid chromatography. The separation conditions were carefully optimized including stationary phases, additives, backpressure, and temperature. Compared to high-performance liquid chromatography, the use of supercritical fluid chromatography could provide a 13 times faster separation. Subsequently, the method was validated and applied for the determination of eight alkaloids from different parts of Mahonia bealei (Fort.) Carr. (stem, root, leaf, and seed). The results indicated a good repeatability with relative standard deviations for overall precisions lower than 3.2%. The limit of detection was between 0.4 and 2.3 μg/mL while limit of quantitation ranged from 1.5 to 7.5 μg/mL. Recovery ranged from 95.7 to 102.5% indicating a validity of recovery. The content of total eight alkaloids was the highest in stem (66.0 μg/g) and root (65.1 μg/g) compared to leaf or seed. Moreover, anti-acetylcholinesterase activity for those extracts was evaluated by Ellman's colorimetric assay. As a result, the acetylcholinesterase inhibitory activity of the extracted samples was in the following decreasing order: stem > root > leaf or seed. In conclusion, the results indicated that supercritical fluid chromatography could be a useful tool for quality control of Mahonia bealei (Fort.) Carr. containing alkaloids as active compounds.

Ultra high efficiency/low pressure supercritical fluid chromatography (UHE/LP-SFC) for triglyceride analysis: Identification, quantification, and classification of vegetableoils.

Triglycerides of vegetable oils have been extensively studied. Non-aqueous reversed-phase liquid chromatography and silver-ion chromatography are most frequently used to achieve their separation. In previous works, we presented the use of supercritical fluid chromatography with long columns (75cm) packed with fused-core particles to provide ultra-high-performance separations, with a low-toxicity fluid (carbon dioxide) compared to the usual liquid-phase methods. In the present paper, we describe the quantification of triglycerides with supercritical fluid chromatography and evaporative light-scattering detection. Thanks to the isocratic elution mode, this quantification can be simplified, assuming (a) identical response coefficients for compounds having a close structure, (as only triglycerides are quantified), and (b) constancy of the response coefficient along the analysis (no elution gradient). Therefore, the relative concentrations of triglycerides were easily assessed. Only one calibration curve for one reference compound (in this case triolein) was required. The resulting relative concentrations are in good accordance with the numerous publications available. Relative quantification with UV detection at 210nm is also proposed, facilitated by the very low UV absorption of carbon dioxide and with a calibration curve taking account of the variation of UV response according to double bond number. Nineteen vegetable oils are compared. The identification of triglycerides was carried out based on previous knowledge of these oils, but also with the help of a Goiffon retention diagram, based on the relationship between the logarithm of retention factor and the total double bond number. Finally, cluster analyses were computed, based on evaporative light-scattering detection or UV quantification data. They allow a quick comparison of the triglyceride content between the oils, in the goal to exchange one by the other for certain applications, or to compare a new oil to well-known ones.

The differences in matrix effect between supercritical fluid chromatography and reversed phase liquid chromatography coupled to ESI/MS

For many sample matrices, matrix effects are a troublesome phenomenon using the electrospray ionization source. The increasing use of supercritical fluid chromatography with CO2 in combination with the electrospray ionization source for MS detection is therefore raising questions: is the matrix effect behaving differently using SFC in comparison with reversed phase LC? This was investigated using urine, plasma, influent- and effluent-wastewater as sample matrices. The matrix effect was evaluated using the post-extraction addition method and through post-column infusions. Matrix effect profiles generated from the post-column infusions in combination with time of flight-MS detection provided the most valuable information for the study. The combination of both qualitative and semi-quantitative information with the ability to use HRMS-data for identifying interfering compounds from the same experiment was very useful, and has to the authors' knowledge not been used this way before. The results showed that both LC and SFC are affected by matrix effects, however differently depending on sample matrix. Generally, both suppressions and enhancements were seen, with a higher amount of enhancements for LC, where 65% of all compounds and all sample matrices were enhanced, compared to only 7% for SFC. Several interferences were tentatively identified, with phospholipids, creatinine, and metal ion clusters as examples of important interferences, with different impact depending on chromatographic technique. SFC needs a different strategy for limiting matrix interferences, owing to its almost reverse retention order compared to RPLC.

Evaluation of an amide-based stationary phase for supercritical fluid chromatography.

A relatively new stationary phase containing a polar group embedded in a hydrophobic backbone (i.e., ACE®C18-amide) was evaluated for use in supercritical fluid chromatography. The amide-based column was compared with columns packed with bare silica, C18 silica, and a terminal-amide silica phase. The system was held at supercritical pressure and temperature with a mobile phase composition of CO2 and methanol as cosolvent. The linear solvation energy relationship model was used to evaluate the behavior of these stationary phases, relating the retention factor of selected probes to specific chromatographic interactions. A five-component test mixture, consisting of a group of drug-like molecules was separated isocratically. The results show that the C18 -amide stationary phase provided a combination of interactions contributing to the retention of the probe compounds. The hydrophobic interactions are favorable; however, the electron donating ability of the embedded amide group shows a large positive interaction. Under the chromatographic conditions used, the C18 -amide column was able to provide baseline resolution of all the drug-like probe compounds in a text mixture, while the other columns tested did not.

Rapid chiral separation of atenolol, metoprolol, propranolol and the zwitterionic metoprolol acid using supercritical fluid chromatography–tandem mass spectrometry – Application to wetland microcosms

A method for enantiomeric separation of the three β-blocking agents atenolol, metoprolol, propranolol and the zwitterionic metoprolol acid, a major metabolite of both metoprolol and in environmental matrices also atenolol, has been developed. By use of supercritical fluid chromatography and the polysaccharide-based Chiralpak® IB-3, all four compounds were simultaneously enantiomerically separated (Rs>1.5) within 8min. Detection was performed using tandem mass spectrometry, and to avoid isobaric interference between the co-eluting metoprolol and metoprolol acid, the achiral column Acquity® UPC2 BEH 2-EP was attached ahead of to the chiral column. Carbon dioxide with 18% methanol containing 0.5% (v/v) of the additives trifluoroacetic acid and ammonia in a 2:1 molar ratio were used as mobile phase. A post column make-up flow (0.3mL/min) of methanol containing 0.1% (v/v) formic acid was used to enhance the positive electrospray ionization. Detection was carried out using a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode, using one transition per analyte and internal standard. The method was successfully applied for monitoring the enantiomeric fraction change over time in a laboratory scale wetland degradation study. It showed good precision, recovery, sensitivity and low effect of the sample matrix.

Supercritical Fluid Chromatography--Theoretical Background and Applications on Natural Products.

The use of supercritical fluid chromatography for natural product analysis as well as underlying theoretical mechanisms and instrumental requirements are summarized in this review. A short introduction focusing on the historical development of this interesting separation technique is followed by remarks on the current instrumental design, also describing possible detection modes and useable stationary phases. The overview on relevant applications is grouped based on their basic intention, may it be (semi)preparative or purely analytical. They indicate that supercritical fluid chromatography is still primarily considered for the analysis of nonpolar analytes like carotenoids, fatty acids, or terpenes. The low polarity of supercritical carbon dioxide, which is used with modifiers almost exclusively as a mobile phase today, combined with high efficiency and fast separations might explain the popularity of supercritical fluid chromatography for the analysis of these compounds. Yet, it has been shown that more polar natural products (e.g., xanthones, flavonoids, alkaloids) are separable too, with the same (if not superior) selectivity and reproducibility than established approaches like HPLC or GC.

Method development for impurity profiling in SFC: The selection of a dissimilar set of stationary phases

Supercritical fluid chromatography (SFC) is drawing considerable interest as separation technique in the pharmaceutical industry. The technique is already well established in chiral separations both analytically and on a preparative scale. The use of SFC as a technique for drug impurity profiling is examined here. To define starting conditions in method development for drug impurity profiling, a set of dissimilar stationary phases is screened in parallel. The possibility to select a set of dissimilar columns using the retention factors (k-values) for a set of 64 drugs measured on 27 columns in SFC was examined. Experiments were carried out at a back-pressure of 150bar and 25°C with a mobile phase consisting of CO2 and methanol with 0.1% isopropylamine (5–40% over 10min) at a flow rate of 3mL/min. These k-values were then used to calculate correlation coefficients on the one hand and to perform a principal component analysis on the other. The Kennard and Stone algorithm, besides dendrograms and correlation-coefficient colour maps were used to select a set of 6 dissimilar stationary phases. The stationary phase characterization results from this study were compared to those from previous studies found in the literature. Retention mechanisms for compounds possessing different properties were also evaluated. The dissimilarity of the selected subset of 6 stationary phases was validated using mixtures of compounds with similar properties and structures, as one can expect in a drug impurity profile.

Prospects for petroleum mass spectrometry and chromatography

Several recently developed analytical techniques, based on high-end mass spectrometry and chromatography, for dealing with challenges in petroleum characterization are reported. Folded flight path time-of-flight mass spectrometry provides resolving power up to 100000, enabling accurate mass measurement for molecular formula determination with high confidence. Atmospheric pressure chemical ionization (APCI) can be used in both gas chromatography (GC, as APGC) and liquid chromatography (LC) for analyzing non-polar hydrocarbons as well as polar compounds. The improvement in chromatography facilitates the mass spectrometric analysis through online coupling. Comprehensive two-dimensional gas chromatography (GC×GC) resolves overlapping components, rendering accurate identification and quantitation. Supercritical fluid extraction has been developed as an alternative method to replace traditional solvent extraction methods and eliminate the use of large volumes of solvents that can be harmful to health and environment. Supercritical fluid chromatography (SFC) has been developed as a convergence of GC and LC chromatographic techniques. The use of SFC for heavy oils and residua has been demonstrated. Prospective developments in the use of mass spectrometric and chromatographic methods for petroleum characterization are also described.

Effects of selected parameters on the response of the evaporative light scattering detector in supercritical fluid chromatography

Due to the renewal of the use of supercritical fluid chromatography (SFC), the coupling of SFC with evaporative light scattering detection (ELSD) needs to be revisited. Indeed, SFC and ELSD apparatus have evolved, while understanding of the nebulisation process has improved. This detector, first developed for polymer analyses can be used for a lot of compounds that do not display sufficient UV absorption. Because the response of ELSD is related to numerous parameters, such as mobile phase composition, the velocity difference between the mobile phase and the nebulisation gas, or the nebulisation temperature, many additional studies are necessary to understand its behaviour in SFC as compared to HPLC. The effects of the nature of co-solvent added to carbon dioxide (methanol, ethanol and acetonitrile), the injected volume and the flow rate on the ELSD response were studied. Certain parameters induce great changes on the nebulisation recovery (the proportion of mobile phase entering the drift tube), allowing for a dramatic improvement in peak area. Moreover, effects of the particle size in the aerosol were also observed, related, as expected, to the modifier nature, but strangely also to the injected volume. Specific and positive behaviour of the ELSD detection on the apparent peak efficiency was confirmed in SFC, for large injected volumes. Finally, a flow rate increase reduces peak area, showing that the use of high flow rates, although attractive to shorten analysis duration, is not favourable to ELSD response. Numerous parameters acting on detector response are modified by the flow rate change, such as the mobile phase velocity. Moreover, measurement of the nebulisation chamber temperature shows the strong decrease of temperature for high flow rates, due to the supercritical fluid depressurisation.

Supercritical fluid chromatography for the enantioseparation of pharmaceuticals

Chirality has a significant impact on drug discovery and development processes in the pharmaceutical industry. As the number of enantiopure drugs launched onto the market is yearly increasing, the need for fast and performant enantioseparation methods with minimal costs is becoming more compelling. In this context, sub- and supercritical fluid chromatography (SFC), being applicable at an analytical, as well as at a preparative scale is gaining more interest. In this review a practical overview is given of the different parameters that are important in supercritical fluid chromatographic separations. A comparison is made between the applicability for chiral separations of SFC and conventional high-performance liquid chromatography (HPLC), and illustrated by means of examples. Different aspects of method development and the upscaling feasibility in SFC are discussed. This review aims to give the reader a practical insight in the use of supercritical fluid chromatography for the chiral separation of pharmaceutical compounds.

Orthogonality of SFC versus HPLC for Small Molecule Library Separation

Preparative HPLC and HPLC-MS are well established as the methods of choice for purification of pharmaceutical library compounds. Recent advances in supercritical fluid chromatography (SFC) have now made SFC a viable alternative to HPLC for this application. One of the potential arguments for using SFC in place of, or in addition to, HPLC is that it may offer different selectivity and thus has the potential for improved separation success rates. In this paper, we examine relative success rates for SFC and HPLC in obtaining adequate selectivity for successful separation. Our results suggest that use of SFC in addition to HPLC may result in a slight (1-2%) improvement in success rate compared to use of HPLC alone.

Supercritical fluid chromatography comparison of the poly( trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column with several derivatized polysaccharide-based stationary phases

The poly( trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column has so far been primarily used with normal phase and polar organic mobile phase chromatography. Its use in supercritical fluid chromatography (SFC) was investigated via the analysis of 40 commercial and 100 proprietary compounds using a 12-min gradient with methanol as a modifier. Results were then compared against those obtained from the popular derivatized polysaccharide-based chiral stationary phases (CSPs) such as Chiralpak AD-H and Chiralpak AS-H as well as Chiralcel OD-H and Chiralcel OJ-H columns. P-CAP demonstrated separation of 25% of the 140 total compounds, while each of the derivatized polysaccharide-based CSPs separated at least 46%. A study that compared the loading of 1,1′-bi-2-naphthol with P-CAP and Chiralpak AS columns indicated a similar trend in resolution vs. amount injected, though AS appeared capable of allowing a greater loading of material. The P-CAP column was found to be beneficial in the separation of a complex mixture of enantiomers and achiral impurities, where the derivatized polysaccharide-based columns did not show as desirable of a separation. A key advantage of this type of chiral stationary phase is the fact that it is available in both enantiomeric forms, allowing manipulation of elution order of enantiomers, which is especially helpful for preparative applications. P-CAP also demonstrated that it could resolve an achiral impurity from the desired compound in a different mixture, while the same impurity co-eluted on the Chiralpak AD-H column. Overall, the synthetic polymer-based P-CAP showed less chiral discrimination power compared to the derivatized polysaccharide-based CSPs under the conditions explored in this study.

Application of supercritical fluid chromatography to the analysis of hydrophobic metabolites

This review describes the usefulness of supercritical fluid chromatography (SFC) in the analysis of hydrophobic metabolites. The use of SFC for the analysis of naturally occurring polyprenols markedly improves the chromatographic resolution of polyprenol homologues and their geometric isomers as compared to conventional HPLC. Under optimized SFC conditions, individual homologues with 10-100-mers were separated. Furthermore, we established an analytical system for the fingerprinting and profiling of diverse lipids through the usage of SFC-MS. When a cyanopropylated silica gel packed column was used for the separation, 14 lipids were successfully detected, and the time required for analysis was less than 15 min. The use of an octadecylsilylated column for the separation depended on the differences in the fatty acid side chains. SFC is a useful separation technology for hydrophobic metabolites, which are difficult to be separated by HPLC.

Simulated moving columns technique for enantioselective supercritical fluid chromatography

This article describes a very useful extension of an unique column switching technique called "Simulated Moving Columns" (SMC) that was previously reported for chiral high performance liquid chromatography (HPLC) (Zhang and McConnell, Journal of Chromatography A 2004;1028:227-238). SMC uses two or three short chiral columns connected in series, and enables the unresolved enantiomers to separate repeatedly and exclusively through each of the columns until sufficient resolution is attained. The technique is significantly enhanced through the use of supercritical fluid chromatography (SFC). The supercritical or near critical carbon dioxide (CO(2)) used in the mobile phase of SFC possesses the properties of a liquid as well as a gas, and usually results in much sharper peaks compared to HPLC. Consequently, by combining SMC with SFC (SMC-SFC), we were able to dramatically increase the number of SMC cycles with significantly less band broadening compared to HPLC. For the first time, an enantioselective SFC separation was demonstrated by increasing the column from the actual 20 cm length to reach a half meter virtual length with remarkably enhanced efficiency. Off-column band broadening resulting from a two-column SMC system was measured, and its impact on the enantioselectivity of SMC-SFC was found to be much less than in SMC-HPLC.

超臨界流体クロマトグラフィーを用いた疎水性代謝物の分離分析

In this paper, we describe the usefulness of supercritical fluid chromatography (SFC) in the analysis of hydrophobic metabolites. The use of SFC in the analysis of plant polyprenols markedly improved the chromatographic resolution of polyprenol homologs and their geometric isomers, compared with conventional high-performance liquid chromatography. Individual homologs from 10 to 100 mers were separated under optimized SFC conditions. Then, we established an analytical system for fingerprinting and profiling of diverse lipids using supercritical fluid chromatography/mass spectrometry (SFC/MS). When a cyanopropylated silica gel packed column was used for the separation, 14 lipids were successfully detected, and the analysis time was less than 15 min. The use of an octadecylsilylated column resulted in separation, depending on the differences in the unsaturation of the fatty acid side chains and isomer separation.